This invention relates to the new protein, cartelin, which inhibits in vivo and in vitro polymorphonuclear neutrophils (PMN) elastase. More particularly, the invention relates to the use of the new protein in human therapeutics of elastase-mediated tissue injury disease.
In these diseases, metastatic cells or inflammatory cells, such as leucocytes, diffuse in tissue. PMN elastase and its cohort enzymes have a broad specificity towards endogenous substrates, including amorphous elastic and connective tissue components, such as collagen type III, which is the supporting component of lung, blood vessel, and gut connecting tissue, collagen type IV, which is important for the integrity of epithelial and endothelial basement membrane, and fibronectin, which is the cell-adhesion molecule.
These related diseases include pulmonary emphysema, chronic bronchitis, cystic fibrosis, bronchiectasis, adult respiratory distress syndrome, atheriosclerosis, arthritis, psoriasis, vasculitis, glomerulonephritis, consumption coagulopathies associated with gram-negative sepsis, and leukemias. Furthermore, the invasion of tissue during metastasis by neoplastic cells, which is accompanied by collagen cleavage, would be controlled by elastase inhibition.
It has been noticed that some cartilage intrinsically retards the invasion of tumor cells and leucocytes. The resistance is in part due to the avascular nature, the organization, and the composition of the extracellular matrix.
This natural defense against invasion appears to be contained in the extractable cartilage matrix compound named antiinvasive-factor (AIF). See Bone Metastasis, G. K. Hall, Medical Publishers, Boston, Mass., 1981, pp. 153-154, incorporated herein by reference. Within this mixture of proteins are a number of protease inhibitors and growth regulatory factors which have been partially purified.
Initial examination of AIF shows that it contains inhibitory activity toward leucocyte-elastase. Therefore, it has been hypothesized that this inhibitor may be an agent responsible for natural protection against metastasis.
In the present invention, AIF was examined for PMN-elastase inhibitory activity using analytical and preparative chromatographic and electrophoretic techniques. The purification steps were monitored by measuring the inhibiting activity with a leucocyte elastase test using a chromogen substrate. A specific PMN-elastase inhibitor was isolated and characterized as a protein of 15,000 daltons on SDS-PAGE with a high cationic character (pI (isoelectric point) greater than 9.5) and a strong hydrophobic behavior.
The amino-terminal-sequence and the sequence of a tryptic peptide showed no homology to other proteins or protease inhibitors or their nucleotide translations. The detected sequences are highly characteristic for the new inhibitors and therefore can be used to synthesize hybridization probes to screen a cDNA mammalian chondrocyte library and clone the gene.